The goal of this proposal is to further characterize the functional activities of MEF, a member of the ETS family of transcription factors, in hematopoietic cells. We have learned a great deal about the function of MEF, having identified its essential role in innate immunity and in regulating hematopoietic stem cell quiescence. Our focus now is to more completely define the role of MEF in controlling normal (and leukemic) hematopoietic stem cell behavior, characterize the cell type specificity of its transforming properties, and understand the mechanistic basis for its growth promoting effects. To accomplish this, we propose to further define the role of MEF in regulating hematopoietic stem cell (HSC) quiescence and the chemo-sensitivity and radio-sensitivity of hematopoietic cells, by modulating MEF levels in HSCs ( by both overexpressing MEF and reducing the level of MEF using siRNA). We will also define the role that changes in cyclin C expression (or cyclin D2 or D3) play in the regulation of quiescence by MEF, and will define how known cell cycle regulatory genes, such as p21, modulate the effects of MEF on HSC behavior. We will also further define the role of MEF in malignant transformation by defining the importance of MEF in the transforming properties of known oncogenes (such as H-RasV12) using mouse embryonic fibroblasts, and by determining how changes in MEF levels cooperate or antagonize the ability of leukemia-associated oncogenes such as BCR-ABL, NUP98-HOXA9, and NUP98-HOXD13 to transform hematopoietic cells. We will also define the role MEF plays in regulating p53 dependent processes in the cell by carefully defining the phenotype of MEF null, p53 null double knock-out mice. To define the mechanisms underlying these effects of MEF on cell behavior, we will determine how phosphorylation of MEF by cellular kinases (e.g. the ERK2 MAP kinase) affects the function of MEF in a variety of biological assays, and will utilize transcript profiling to further define which MEF target genes are essential for its effects on cellular quiescence, growth, and transformation. These studies will provide valuable insight into the transcriptional regulation of normal and leukemic hematopoietic stem cell quiescence, and cellular transformation, which are key issues in stem cell biology and the pathogenesis of human cancer.